With modern requirements for the control of absolute configuration in drug molecules and for their environmentally responsible and cost-effective production, enzymes have become increasingly valuable catalysts for the large-scale synthesis of pharmaceutical intermediates. However, enzymes are sensitive to elevated temperatures and the presence of co-solvents, and are often not optimal in terms of rate, lifetime, product inhibition, and substrate acceptance. The standard ways to ameliorate these problems are to immobilize enzymes on solid supports to improve their batch recyclability, and to improve the properties of enzymes by directed evolution. Both techniques require much time and effort on a case-by-case basis. We have developed a method to incorporate multiple copies of active enzymes inside the very stable protein shell of the bacteriophage QB capsid. These particles can be produced and isolated in large quantities, are extraordinarily stable against denaturation and protease digestion, and impart additional stability to the proteins packaged inside. When enzyme substrates and products can diffuse through the large pores in the capsid structure, the enzymes entrained within can exhibit high catalytic activities. Qapsule Technologies, Inc. proposes to develop such particles for the synthesis of high-value-added pharmaceutical intermediates. This Phase I proposal identifies two proof-of-concept targets - an aldolase for asymmetric catalysis and a fluorinase for the installation of fluorine atoms, both highly prized by the pharmaceutical industry. We will demonstrate the high-yield production of nanoparticles containing multiple copies of these enzymes, determine their catalytic activities, and test their stabilities, activities, lifetime, and process characteristics relative to non-encapsulated analogues. Clear benchmarks for success are identified, the achievement of which will set the stage for Phase II development and the initiation of a business plan to bring these capabilities to market. Our technology for enzymatic production and packaging can be universally applied to almost any enzyme of interest. It therefore adds to efforts of directed evolution and provides a constant platform that will streamline production, use, and workup of biocatalytic engines for large-scale pharmaceutical synthesis. PUBLIC HEALTH RELEVANCE: Enzymes are the catalysts of choice for many transformations used in the synthesis of pharmaceutical intermediates, but are often too unstable for efficient use. We will develop robust protein nanoparticles containing functional enzymes, which stabilize these catalysts and allow for convenient production and processing.